Bacterial small noncoding RNAs (sRNAs) play diverse roles as gene expression regulators in a variety of physiological processes. Generally, bacterial sRNAs modulate translation activity and/or endogenous stability of their target mRNAs via RNA-RNA base pairing. In Escherichia coli (E. coli), about a hundred species of sRNAs have been experimentally identified, and nearly half of these sRNAs have been functionally analyzed.
The majority of identified sRNAs is encoded in trans at different chromosomal locations from target genes, and usually suppresses target gene expression by inhibiting translation or inducing mRNA degradation with the aid of an abundant RNA chaperone protein, Hfq. Other sRNAs are encoded in cis on strands opposite their target. Unlike trans-encoded sRNAs, cis-encoded sRNAs have extensive regions capable of perfect base-pairing, but the actual base-pairing regions appear limited to specific sequences. Additionally, their functions are generally independent of Hfq.
Several researchers have attempted to design strategies for silencing or knockdown of specific genes by mimicking the general functions of sRNAs. In eukaryotes, induction of specific gene silencing by short interfering RNAs (siRNAs) has been extensively used for elucidating gene function and developing therapeutic agents. Similarly, gene silencing in bacteria could be achieved using artificial sRNA (afsRNA) loaded with well-defined target recognition sequences. To design effective afsRNAs in E. coli, factors that affect interactions with target mRNA should be considered, including accessibility of target site, base-pairing energy, secondary structures of target mRNA and afsRNA, off-target effects of afsRNA, and reaction kinetics.
That is, knockdown or silencing of a specific gene expression can be very effectively used for elucidating gene function. To date, efficient silencing methods have been established in eukaryotic cells, but not in bacteria. Accordingly, there is a demand for a new strategy for knockdown or silencing of specific genes in bacteria by using afsRNAs.